Construction of Hansenula polymorpha strains with improved thermotolerance

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high‐temperature resistance and high‐temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat‐shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12‐fold increased tolerance to heat‐shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8‐fold improvement of ethanol production from xylose at 50°C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L⁻¹, our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high‐temperature ethanol producers from this pentose. Biotechnol. Bioeng. 2009; 104: 911–919. © 2009 Wiley Periodicals, Inc.     

Hybridization of asporogenic strains of Hansenula polymorpha yeasts by protoplast fusion

Intrastrain and interstrain hybrids of different ploidy were produced by the fusion of protoplasts of Hansenula polymorpha haploid and diploid strains. The diploid hybrids were found to be stable in contrast to the triploid and tetraploid hybrids. The instability of the triploid and tetraploid states in Hansenula polymorpha was expressed in the elevated frequency of spontaneous formation of auxotrophic markers and in the decreased content of DNA per cell in the course of storage.     

Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha

The Delta(6)-desaturase gene isoform II involved in the formation of gamma-linolenic acid (GLA) was identified from Mucor rouxii. To study the possibility of alteration of the synthetic pathway of essential fatty acids in the methylotrophic yeast, Hansenula polymorpha, the cloned gene of M. rouxii under the control of the methanol oxidase (MOX) promoter of H. polymorpha, was used for genetic modification of this yeast. Changes in flux through the n-3 and n-6 pathways in the transgenic yeast were observed. The proportion of GLA varied dramatically depending on the growth temperature and media composition. This can be explained by the effects of either substrate availability or enzymatic activity. In addition to the potential application for manipulating the fatty acid profile, this study provides an attractive model system of H. polymorpha for investigating the deviation of fatty acid metabolism in eukaryotes.     

Growth simulation of Hansenula polymorpha

Summary Cultivation of Hansenula polymorpha with substrate ethanol in a bench-scale tower loop reactor was simulated by means of a distributed parameter model with regard to the dissolved oxygen and substrate in the medium, oxygen and CO₂ in the gas phase, and a lumped parameter model with regard to the cell mass. Space and time independence of the substrate and oxygen limiting constants of the Monod model, K_S and K_O, was assumed. Time variations of the yield coefficients, Y_X/S and Y_X/O, were allowed for.     

Peroxisome homeostasis in Hansenula polymorpha

Peroxisomes are essential organelles in many eukaryotes. Until recently, the main focus of the investigations concerning these important organelles was to understand the biogenesis of the peroxisome (induction, proliferation and matrix protein import). However, when peroxisomes become redundant they are quickly degraded by highly selective processes known as pexophagy. The first molecular studies on pexophagy have indicated that this process shares many features with certain transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole targeting and endocytosis). Nevertheless, recent data demonstrate that in addition to common genes also unique genes are required for these transport processes. The main focus for the future should therefore be on identifying the unique determinants of pexophagy. Earlier results suggest that in the methylotrophic yeast Hansenula polymorpha proteins located on the peroxisome itself are required for pexophagy. Thus, it has become essential to study in detail the role of peroxisomal membrane proteins in the degradation process. This review highlights the main achievements of the last few years, with emphasis on H. polymorpha.     

Novel genetic tools for Hansenula polymorpha

Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H.?polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H.?polymorpha. For this, we generated a yku80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H.?polymorpha.     

多形汉逊酵母研究进展

作为研究甲醇代谢、过氧化物酶体稳态和硝酸盐吸收的模式生物,多形汉逊酵母近年来在基础研究领域日益受到重视。在工程应用领域,利用多形汉逊酵母表达真核外源基因有特殊的优势。譬如容易得到高拷贝,在含油酸的培养条件下能够表达膜蛋白等。已有多种外源蛋白在多形汉逊酵母系统中得到表达。本文综述了多形汉逊酵母的基本生物学性质、基础研究领域概况及其在外源基因表达方面的特点和进展。     

Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732

Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.     

Determining a carbohydrate profile for Hansenula polymorpha

The determination of the levels of carbohydrates in the yeast Hansenula polymorpha required the development of new analytical procedures. Existing fractionation and analytical methods were adapted to deal with the problems involved with the lysis of whole cells. Using these new procedures, the complete carbohydrate profiles of H. polymorpha and selected mutant strains were determined and shown to correlate favourably with previously published results.     

Specific features of telomerase RNA from Hansenula polymorpha

Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcribes the template beyond the predicted boundary and adds a nontelomeric dT in vitro. Sequencing of the chromosomal ends revealed that this nucleotide is specifically present as a terminal nucleotide at the 3′ end of telomeres. Mutational analysis of HpTER confirmed that the incorporation of dT functions to limit telomere length in this species.     

Cold-inducible selective degradation of peroxisomes in Hansenula polymorpha

Exposure of Hansenula polymorpha cells, grown in batch cultures on methanol at 37 degrees C, to a cold treatment (18 degrees C) is paralleled by a rapid degradation of peroxisomes present in these cells. Remarkably, the events accompanying organelle degradation at 18 degrees C are similar to those of selective glucose-induced peroxisome degradation in wild-type cells, described before. This observation was strengthened by the finding that cold-induced peroxisome degradation was not observed in mutants impaired in selective peroxisome degradation (Atg(-) mutants). Biochemical data indicated that the onset of peroxisome degradation was not triggered by the inactivation of peroxisome function due to the fall in temperature. We show that our findings have implications in case of fluorescence microscopy studies that are generally not conducted at physiological temperatures and thus may lead to strong morphological alterations unless proper precautions are taken.     

Cultivation of Hansenula polymorpha in tower loop reactors

Summary Hansenula polymorpha was cultured for extended periods in an air lift tower loop reactor (15 cm in diameter with a 275 cm bubbling layer height) with ethanol and/or glucose as the substrates. At constant operation conditions variations of the following parameters were measured: the consumption of the substrate and oxygen, the production of CO₂ and biomass, the longitudinal concentration profile of dissolved oxygen, the oxygen and substrate yield coefficients, the respiratory quotient and the specific interfacial area and volumetric mass transfer coefficient. The influence of the microorganisms on the oxygen transfer rate is discussed especially in the case of glucose repression.     

High-level expression of foreign genes in Hansenula polymorpha

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.     

Methanol Inhibition in Continuous Culture of Hansenula polymorpha

Growth inhibition of Hansenula polymorpha DL-1 by methanol, formaldehyde, formate, and formic acid was examined to determine the causes of unstable behavior observed during continuous cultures on methanol. The much greater inhibition of growth by formic acid than by formate and the effect of formic acid excretion and assimilation on pH helped to explain culture dynamics observed after transitory oxygen limitations. Oxygen limitation caused by temporary reduction of agitation in a continuous fermentation caused methanol to accumulate to inhibitory concentrations. Immediately after resumption of agitation, formic acid was produced and caused culture inhibition. To ensure the stability of H. polymorpha in continuous culture, it was therefore necessary to prevent transient methanol accumulation.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

Production of catalase-free alcohol oxidase by Hansenula polymorpha

Summary Many of the potential technical applications of alcohol oxidase (MOX; EC 1.1.3.13) are limited by the presence of high activities of catalase in the enzyme preparations. In order to circumvent laborious and costly purification or inactivation procedures, the induction of MOX in a catalase-negative mutant of Hansenula polymorpha has been studied. Emphasis was laid on the induction of activities of MOX and the dissimilatory enzymes in continuous cultures grown on various mixtures of formate/glucose and formaldehyde/glucose. In continuous cultures of the catalase-negative mutant grown on these mixtures, MOX can be induced efficiently. To obtain a stable and productive process, the ratio of the substrates is of critical importance. The optimal ratios of the mixtures for the catalase-negative strain for formate/glucose and formaldehyde/glucose were 3:1 and 1–2:1, respectively. Under identical cultivation conditions the wild-type strain showed similar induction patterns for MOX and the dissimilatory enzymes formaldehyde dehydrogenase (FaDH) and formate dehydrogenase (FoDH). The MOX levels in the catalase-negative strain were approx. 50% of those in the wild-type strain.     

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha . Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system ( K _m for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system ( K _m for glucose 0.05–0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide- m -chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.     

Hansenula polymorpha Pex14p phosphorylated in vivo

Hansenula polymorpha Pex14p is a novel peroxisomal membrane protein essential for peroxisome biogenesis. In vivo labeling experiment of wild-type cells with ³²P-orthophosphate and alkaline phosphatase treatment of labeled Pex14p indicate that Pex14p is phosphorylated in vivo. Analysis of the phosphoamino acid in the phosphorylated Pex14p suggested that the major phosphoamino acid was acid labile. Using expression system of several truncated Pex14ps in a PEX14-deletion strain it is suggested that the phosphorylation site of Pex14p resides in the C-terminal 58 residues.